title: Enzymatic and structural characterization of hydrolysis of gibberellin A4 glucosyl ester by a rice β-D-glucosidase.
abstract: In order to identify a rice gibberellin ester β-D-glucosidase, gibberellin A4 β-D-glucosyl ester (GA4-GE) was synthesized and used to screen rice β-glucosidases. Os3BGlu6 was found to have the highest hydrolysis activity to GA4-GE among five recombinantly expressed rice glycoside hydrolase family GH1 enzymes from different phylogenic clusters. The kinetic parameters of Os3BGlu6 and its mutants E178Q, E178A, E394D, E394Q and M251N for hydrolysis of p-nitrophenyl β-D-glucopyranoside (pNPGlc) and GA4-GE confirmed the roles of the catalytic acid/base and nucleophile for hydrolysis of both substrates and suggested M251 contributes to binding hydrophobic aglycones. The activities of the Os3BGlu6 E178Q and E178A acid/base mutants were rescued by azide, which they transglucosylate to produce β-D-glucopyranosyl azide, in a pH-dependent manner, while acetate also rescued Os3BGlu6 E178A at low pH. High concentrations of sodium azide (200-400 mM) inhibited Os3BGlu6 E178Q but not Os3BGlu6 E178A. The structures of Os3BGlu6 E178Q crystallized with either GA4-GE or pNPGlc had a native α-D-glucosyl moiety covalently linked to the catalytic nucleophile, E394, which showed the hydrogen bonding to the 2-hydroxyl in the covalent intermediate. These data suggest that a GH1 β-glucosidase uses the same retaining catalytic mechanism to hydrolyze 1-O-acyl glucose ester and glucoside.
authors.affiliation: School of Biochemistry and Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima, Thailand.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Hua
authors.forename: Sompong
authors.initials: S
authors.lastname: Sansenya
authors.forename: Chiraporn
authors.initials: C
authors.lastname: Saetang
authors.forename: Shinji
authors.initials: S
authors.lastname: Wakuta
authors.forename: James R
authors.initials: JR
authors.lastname: Ketudat Cairns
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004789:Enzyme Activation; D004795:Enzyme Stability; D004952:Esters; D005875:Gibberellins / Q000737:chemistry*; D006868:Hydrolysis; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D012275:Oryza / Q000201:enzymology*; D011485:Protein Binding; D011487:Protein Conformation; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry*
chemical_list: D004952:Esters; D005875:Gibberellins; C532593:gibberellin A4; D001617:beta-Glucosidase
keywords: Covalent intermediate; Gibberellin glucosyl ester; Transglycosylation; X-ray crystal structure; β-Glucosidase